This thesis includes three stages of experiments. The goal of the thesis was to prepare nanoparticle-encapsulated curcumin for the purpose of drug delivery. The first step was the nanoparticle preparation. The self-assembly of block copolymer (poly(ε- caprolactone)-b-poly(ethylene oxide)) and curcumin was conducted on a gas-liquid two phase microfluidic reactor. During preparation, various chemical parameters and flow rates were tested. The nanoparticles showed flow variability; the size decreased and the loading efficiency increased with increased flow rates. Increasing the water content and drug-to-polymer loading ratio also proved to increase loading efficiency and decrease the size of the nanoparticles. The release profiles, however, showed fast release rates under various preparation conditions, with a nearly complete release after ~5 h. In the next stage of the research, we considered release optimization in preparation for future pharmacokinetic studies. Increasing the flow rate had a greater influence on slowing down release rates than changing other parameters, such as decreasing the drug-to- polymer loading ratio or increasing the water content. A procedure to extract and quantify curcumin from mouse blood was also developed in this stage. In the final stage of the research, nanoparticle-encapsulated curcumin was tested on a human breast cancer cell line, MDA-MB-231. The result showed that the nanoparticle formulation had a growth inhibition effect on MDA-MB-231, although the cytotoxicity was compromised by encapsulation in the nanoparticles. / Graduate / 2019-01-13

A study to develop novel lipophilic metal ion complexes based on dihydrocholesterol was undertaken. Steroid ligands functionalised at the 2- and 3- positions were synthesized as possible bidentate ligands for complexation of metal ions. Condensation of 5α-cholestan-3-one with ethyl formate in the presence of base gave 2-hydroxymethylene-5α-cholestan-3-one, and 2- acetyl-5α-cholestan-3-one was obtained by the reaction between 3- trimethylsilyloxy-5α-cholest-2-ene and acetyl chloride. Attempts to synthesize 2,3-dioximino-5α-cholestane from 5α-cholestan-3-one and 2α-hydroxy-5α-cholestan-3-one were unsuccessful. Likewise 2- methylene-5α-cholestan-3-one, which was expected to lead to other bidentate ligands, could not be prepared satisfactorily from 5α-cholestan- 3-one or 3-trimethylsilyloxy-5α-cholest-2-ene.

Utilisation de nanoparticules pour délivrer des protéines dans les épithéliums respiratoires. Caractérisation des mécanismes impliqués. L’administration de médicaments par les voies respiratoires humaines est un domaine de la recherche en pleine expansion. Un effort croissant est porté sur le développement de systèmes innovants capables d’échapper aux mécanismes de clairance des voies respiratoires, d’améliorer la biodisponibilité des molécules d’intérêt, leur absorption dans la muqueuse et leur efficacité thérapeutique. Dans ce contexte, le but de ce travail était d’évaluer le potentiel de nanoparticules polysaccharidiques cationiques et poreuses (NP+) comme vecteurs de protéines à travers les voies respiratoires humaines. Les NP+ sont utilisées avec succès in vivo, comme vecteurs mucosaux dans de nombreuses applications telles que la vaccination, l’allergie, la thérapie anticancéreuse et la délivrance de molécules thérapeutiques. Cependant, les mécanismes d’interaction de ces nanoparticules avec les cellules épithéliales des voies respiratoires sont peu connus. Nous avons étudié l’endocytose, l’exocytose et la localisation intracellulaire de ces nanoparticules dans des cellules épithéliales bronchiques humaines. Leur toxicité a été évaluée sur ces cellules et plus particulièrement leur cytotoxicité et leur génotoxicité. Enfin, nous avons étudié et caractérisé les mécanismes de délivrance intracellulaire de protéines par ces nanoparticules et l’influence de leur composition interne sur ces mécanismes. Nos travaux montrent une endocytose rapide des NP+ par la voie des clathrines, ainsi qu’une importante exocytose par des mécanismes dépendant du cholestérol. Elles sont localisées dans les vésicules de clathrines, les endosomes précoces et pas dans les endosomes tardifs, ni dans les lysosomes. De manière ces nanoparticules s’associent quantitativement aux protéines et augmentent leur délivrance intracellulaire, tout en les protégeant de l’hydrolyse enzymatique à pH physiologique. De plus, la présence de lipides anioniques dans leur structure interne influence significativement les mécanismes d’interactions avec les cellules et de délivrance intracellulaire de protéines. Enfin, les études de toxicité ne montrent aucune cytotoxicité ni génotoxicité à des concentrations < 326 µg/cm2. Ces concentrations sont toutefois très élevées et difficilement atteignables in vivo. En conclusion, les NP+ ne sont pas toxiques sur les cellules épithéliales des voies respiratoires, elles interagissant fortement avec celles-ci et augmentent significativement la délivrance de protéines. Ce travail souligne l’intérêt de développer ce type de nanoparticules pour la délivrance de molécules d’intérêt pharmaceutique à travers les voies respiratoires humaines. / Drug delivery through the human respiratory tract is a promising field under investigation. A growing effort is focused on developing innovative delivery systems able to escape the clearance mechanisms of the respiratory tract, to improve molecules bioavailability, their absorption and their therapeutic efficacy, in the respiratory mucosa. In this context, the aim of this study was to evaluate the potential of polysaccharide cationic porous nanoparticles (NP+) as airway vectors for proteins. NP+ are successfully used as mucosal vectors in vivo, in many applications, including vaccination, allergy, cancer therapy and drug delivery. However, the mechanisms of NP+ interaction with airway epithelial cells remain poorly understood. We investigated the endocytosis, the exocytosis and the intracellular localization of NP+ in human bronchial epithelial cells. We assessed their toxicity on these cells, and particularly their cyto- and genotoxicity. Finally, we studied and characterized the mechanisms of intracellular delivery of proteins by these nanoparticles, and the influence of their inner composition, on these mechanisms. Our results showed a rapid uptake of NP+ via the clathrin endocytosis pathway, and a significant exocytosis via a cholesterol-dependent mechanism. Moreover, NP+ were located in clathrin vesicles, early endosomes but not in late endosomes nor lysosomes. Interestingly, these nanoparticles quantitatively associated proteins and increased their intracellular delivery, while protecting them from enzymatic degradation at physiological pH. Moreover, the presence of anionic lipids in their inner structure significantly influences their interaction with cells and the mechanisms of intracellular delivery. Finally, toxicity studies show no genotoxicity or cytotoxicity of these nanoparticles at concentrations below 326&#956;g/cm². However, these concentrations are very high and hardly realistic in vivo. In summary, NP+ are not toxic to airway epithelial cells, they strongly interact with these cells and significantly increase protein delivery. This work highlights the importance of developing this type of nanoparticles to deliver molecules via the human respiratory tract.

No description available.

Cell penetrating peptides (CPPs) have been used in many areas of drug delivery for mediating the delivery of peptides, protein, DNA, siRNA and liposomes. Additionally they have shown an ability to overcome drug resistance in cells enhancing chemotherapeutic activity. Our group has recently designed a promising multifunctional peptide, Tat-LK15, originating from the fusion of Tat peptide (49-57) with the synthetic amphipathic peptide, LK15. TAT-LK15 was found to be efficient at mediating DNA and siRNA delivery to cells especially contributing to the silencing of bcr-abl oncoprotein over a long period of time. To date, Tat-LK15 peptide’s cells uptake mechanism, which is expected to be influenced by the presence of the amphipathic sequence LK15, which is known to be lytic, has not been assessed. The aim of this project is to study the cellular uptake of the Tat-LK15 peptide and any influence it exerts on the uptake of p-glycoproteins substrates. Also we aimed to explore whether the peptide characteristics could be exploited to improve the delivery of the cytotoxic agent, doxorubicin.Flow cytometry and confocal microscopy experiments revealed that Tat-LK15 uptake was dependent on two mechanisms depending on the concentration of peptide used. At low concentrations (2.5µM and below) the uptake of TAMRA-Tat-LK15 peptide appeared to be temperature-dependent and was inhibited by sodium azide suggesting endocytosis as the main route of entry in the studied cell lines. At 5µM and above, the peptide’s uptake was less reliant on temperature and not be inhibited by sodium azide, but relied on membrane potential. Interestingly in K562 cells, the peptide accumulated on the cell membrane.The Tat-LK15 peptide membrane activity was also characterised: Tat-LK15 lytic activity was concentration-dependent. At 5µM the Tat-LK15 peptide led to approximately 60% LDH release in HT29 cells which is likely to be related to the amphipathic sequence, LK15 renowned for its membrane lytic activity. Further to this, the uptake of a membrane impermeant dye (SYTOX® blue) incubated with the peptide was followed as a function of time in K562 cells with a high content screening. The results appeared to substantiate the above findings that Tat-LK15 peptide has a concentration- and time-dependent membrane lytic activity. An MTT assay, after four hours of incubation, indicated very limited variation in cytotoxicity (EC50 ~ 5µM) over six different cell lines. More importantly, an Annexin V assay suggested a possible induction of apoptosis in the MCF-7 cell line, in contrast a Tat-LK15 concentration-dependent necrosis was observed in a K562 cell line. Finally attempts were made to exploit the properties of Tat-LK15 to overcome drug resistance of doxorubicin. Firstly Tat-LK15 peptide co-incubation with a model P-gp substrate (calcein AM) revealed a significant reduction of calcein fluorescence in K562 and HT29 cells and their doxorubicin resistant sublines (p<0.05). Attempts to conjugate doxorubicin to TAT-LK15 proved difficult. Consequently doxorubicin/Tat-LK15 admixtures were used to study whether the P-gp efflux of doxorubicin in doxorubicin resistance sublines could be circumvented. A significant increase of the toxicity relative to doxorubicin alone for long incubation times on several cell lines (p<0.05) indicated that using TAT-LK15 as an additive could potentially be considered as a drug delivery strategy. Thus the concept of conjugating a well known cell penetrating peptide to an amphipathic sequence is worthwhile however the findings in this study are not sufficient to highlight the full potential and major improvements in efficacy needs to be achieved.

archives@tulane.edu / Glaucoma is a debilitating and insidious disease and is the world’s leading cause of irreversible blindness. There have been many proposed innovations in the ophthalmology space though few have successfully been implemented in humans. The gap between proof of concept studies and market launch has been termed the “valley of death.” The Blake, Ayyala, and John research group have been endeavoring to bring two drug delivery systems through this “valley of death” for the last ten years. These products aim to solve a common problem in glaucoma surgery: post-surgical fibrosis resulting in the need for revision surgery. The two drug systems are a poly(hydroxyl ethyl methacrylate) hydrogel loaded with mitomycin C and a biodegradable poly(lactic-co-glycolic acid) matrix loaded with 5-fluorouracil and mitomycin C. These anti-fibrotics, when released into the surgical site, successfully reduced scar tissue formation in animal models. To translate these technologies to market, we created methods to interrogate their synthesis, studied their properties after sterilization, and performed longitudinal studies to determine their stability. For the pHEMA-based drug delivery system, we introduced a new casting method and compared it to previous studies. This new method reduced casting time two-fold and increased lot-to-lot reproducibility. We also developed an assay for quantifying the amount of drug loaded into each hydrogel. Using this assay, we reduced the loading time of the hydrogels two-fold by more than 5 days. The product was then gamma and e-beam sterilized to determine how sterilization would affect the hydrogel. We showed that the hydrogel releases mitomycin C more slowly after gamma irradiation than after e-beam and that both releases were slower than unsterilized material. This indicates that the hydrogel has cross-linked during the sterilization process. For the PLGA-based drug delivery system, we developed a solvent extraction method for quantifying the amount of drug in each piece. We then used this assay to interrogate different steps in the manufacturing process. We discovered the need for a new casting method using a positive displacement pipette. We tested the homogeneity of the 5-fluorouracil within the polymer matrix and discovered that drug distribution in our films was uniform. We ensured that we could reproducibly create lots of these films. Then, we tested the stability of this drug delivery system after gamma irradiation. We performed a longitudinal shelf-life study to see how temperature and the presence of air could affect the system during 3 months of storage. We then lyophilized our product and compared e-beam and gamma sterilization techniques. These studies contributed to an investigational new drug filing with the FDA which is the next milestone for a drug product before first-in-human trials. / 1 / Mitchell Layton Fullerton

Recently, there has been an explosion of interest in developing biopharmaceutical therapeutics for the treatment of life altering conditions. The main issue with the utilization of biopharmaceutical therapeutics is the mode of administration. The Oral Ghost Drug Delivery (OGDD) device could potentially enable the administration of these peptide therapeutics via the oral route and significantly extend the application of these advanced therapeutic compounds.

Chronic pain poses a major concern to modern medicine and is frequently undertreated, causing suffering and disability. Transdermal delivery is the pivot to which analgesic research in drug delivery has centralized especially with the confines of needle phobias and associated pain related to traditional injections, and the existing limitations associated with oral drug delivery. Highlighted within this thesis is the possibility of further developing transdermal drug delivery for chronic pain treatment using an Electro-Modulated Hydrogel- Microneedle array (EMHM) prototype device for the delivery of analgesic medication

One of the most common methods of drug delivery to the anterior segment of the eye is topical application of an ophthalmic solution or suspension. The ophthalmic solution may contain various particle based materials, such as nanoparticles, to control the rate at which the drug is delivered to the eye. The issue with this delivery method is that there are several barriers at the front of the eye. These barriers, which include a high tear film turnover rate and induced lacrimation, reduce the residence time of the drug at the site of administration and result in 95% of the administered drug being removed systemically or via nasolacrimal drainage. Additionally, once the material has left the target location it should degrade in a controlled manner so that it can be safely removed from the body. The current work focuses on the development of polymeric nanoparticles that can serve as a delivery system for ophthalmic drugs. The material proposed for the nanoparticle synthesis is poly(2-hydroxyethyl methacrylate (HEMA)), a polymer with a long history of ophthalmic compatibility. The original nanoparticle formulation was modified to allow for degradation and mucoadhesion. To facilitate degradation, a crosslinker which degrades under ocular conditions was incorporated. A mucoadhesive polymer was incorporated into the particles to enhance the residence time of the particles at the front of the eye. Size and morphology analysis of the final polymer products showed that nano-sized, spherical particles were produced. FTIR spectra demonstrated that the nanoparticles were comprised of poly(HEMA) and that 3-(acrylamido)phenylboronic acid (3AAPBA) was successfully incorporated. Degradation of nanoparticles containing N,N’-bis(acryloyl)cystamine (BAC) after incubation with DL-dithiothreitol (DTT) was confirmed by a decrease in turbidity, measured by absorbance, and through transmission electron microscopy (TEM). Based on zeta potential results, poly(HEMA, BAC, 3AAPBA) samples C3 to C6 were found to be mucoadhesive. Dexamethasone release from poly(HEMA) nanoparticles and poly(HEMA, BAC, 3AAPBA) nanoparticles, loaded with efficiencies of 15.0% ±1.4% and 5.3% ±0.4%, resulted in rate constants of 0.001 and 0.002, and release exponents of 0.607 and 0.586, respectively. The toxicity of the nanoparticles was tested by incubation in the presence of human corneal epithelial cells (HCEC). In the presence of the poly(HEMA), poly(HEMA, BAC), and poly(HEMA, BAC, 3AAPBA) samples the HCEC viability was found to be 123.6% to 182.5%, 88.5% to 111%, and 69.8% to 85.1%, respectively. The viability of HCEC after incubation with poly(HEMA) was significantly higher compared to poly(HEMA, BAC) samples with a dilution factor of 0 and 2. Additionally, the HCEC viability in the presence of poly(HEMA, BAC, 3AAPBA) sample C6 was found to be significantly lower compared to samples C2 and C3 from Table 3. The previously summarized results suggest that the poly(HEMA) based nanoparticles produced in this work have the potential for drug delivery to the front of the eye. / Thesis / Master of Applied Science (MASc)

In recent decades, a variety of nanoparticle drug delivery systems (NP DDS) – nanometer-scaled materials physically or covalently interacting with therapeutics – has been developed to overcome biological barriers, improve the half-life, reduce toxicity, and improve the efficacy of conventional drug delivery. However, many NP DDS fail to translate to the clinic. While this is in part due to immense heterogeneity within many disease types across individuals, the conflicting size and surface chemistries required in the “drug delivery pathway” (i.e. to avoid the clearance mechanisms and unintended tissues in the body, then to reach and specifically enter target tissues) also pose a significant challenge. Recent advances in the field of drug delivery have created size- and surface-switching nanoparticles that overcome biological barriers. For example, large (100 – 200 nm) NPs are adequate at evading corporeal defense mechanisms, while small (< 50 nm) NPs can actively enter cancerous tissue. Further, release profiles of drug-loaded NP DDS must be tailored to stay within a narrow therapeutic window to prevent toxic effects. This thesis highlights the synthesis of “nanoassemblies”, an NP DDS that contains small, drug-loaded starch nanoparticles (SNPs) within a larger nanogel matrix. Nanoassemblies are chemically tuned to reach specific targets via different administration routes (notably, cancerous tissues via systemic administration and brain tissue via intranasal administration). Furthermore, therapeutic-loaded SNPs are released under endogenous (pH, redox) or exogenous (ultrasound) stimuli for disease-specific release kinetics, allowing for deeper penetration into tumor cores or through the nose-to-brain pathway as required. Both the physicochemical characterization of these nanoassemblies as well as in vitro and in vivo experiments have been performed to assess the efficacy of nanoassemblies in biological systems and how they may provide performance improvements over non-assembled SNPs. As such, nanoassemblies show great promise in overcoming complex biological barriers to ultimately improve drug delivery in clinical applications. / Thesis / Doctor of Philosophy (PhD) / Using drugs to treat diseases is not always effective: the drug often does not work or comes with many side effects. A combination of factors prevents promising drugs from working. Most often the drug is either (partially or fully) removed from the body before it reaches the disease, or it improperly enters healthy tissue to cause undesirable responses. Previous research has shown that if drugs are put into nanoparticles, the nanoparticles can better deliver the drug to the correct target. However, conflicting sizes are needed to travel through different parts of the body, making nanoparticle-based drug delivery only of limited effectiveness in humans. This thesis aims to address these issues by creating “nanoassemblies” – nanoparticles with smaller, drug-containing nanoparticles inside of them – that overcome the typical issues with drug delivery. Nanoassemblies are able to switch their size to better reach the target tissue, ultimately leading to more effective and safe treatments.